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Soybean lipoxygenase‐1 (SBLO‐1) catalyzes the oxygenation of linoleic acid to give primarily 13‐hydroperoxy‐9,11‐octadecadienoic acid (13‐HPOD) plus a small amount (10% at pH 7.5) of 9‐hydroperoxy‐10,12‐octadecadienoic acid (9‐HPOD). Replacement of phenylalanine‐557 with valine increases the amount of 9‐HPOD formed to 30–40%. Following earlier suggestions by Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197), our working hypothesis is that 13‐HPOD arises from tail‐first binding with the methyl terminus of linoleic acid near phe‐557, and that replacement of phe‐557, with smaller residues enables linoleic acid to bind in a head‐first manner leading to 9‐HPOD. To test this model, the F557S mutant was prepared, and its activity was determined with linoleic acid and 20‐hydroxy‐5,8,11,14‐eicosatetraenoic acid (20‐HETE), which has a hydroxyl group on the terminal carbon that might form a hydrogen bond with the hydroxyl group of serine‐557. Kinetics studies on the mutant were complicated by pronounced substrate inhibition at high substrate concentration. At low substrate concentration, the activity of the F557S mutant is 25% of wild‐type with linoleic acid as substrate and 65% of wild‐type with 20‐HETE as substrate. Oxygenation of linoleic acid by the F557S mutant produced only 8% of 9‐HPOD, considerably less than the percentage of 9‐HPOD produced by the F557V mutant. This result indicates that the size of the residue at position 557 is not the only factor that affects the regiochemistry of the reaction. To test the hypothesis that 9‐oxygenation results from head‐first binding, linoleoyl‐L‐valine (LLV) and linoleoyl‐D‐valine (LDV) have been investigated as substrates of the F557V mutant. The percentage of 9‐oxygenation with these substrates is comparable to what is observed with linoleic acid and shows little or no dependence on the stereochemistry of the valine moiety. These findings suggest that the 9‐oxygenation of LLV and LDV does not involve head‐first binding.Support or Funding InformationNSF CHE‐1213262